Fibrinolysis and fibrinogenolysis treatment

ABSTRACT

Disclosed is a fibrinolysis and fibrinogenolysis treatment which includes parenterally introducing into the body of a human patient human plasmin in fibrinolytically and fibrinogenolytically active form at a concentration and for a time sufficient to permit fibrinolytically and fibrinogenolytically active human plasmin to reach a concentration about the site of any intravascular clot sufficient to lyse the clot and/or to reduce circulating fibrinogen levels.

RELATED APPLICATION

This application is a division of application Ser. No. 07/755,501, filedAug. 28, 1991, U.S. Pat. No. 5,288,489.

This invention relates to the treatment and prevention of thromboticdisorders.

BACKGROUND OF THE INVENTION

Thromboembolic disease, i.e., blockage of a blood vessel by a bloodclot, affects many adults and can be a cause of death. Mostspontaneously developing vascular obstructions are due to the formationof intravascular blood clots, also known as thrombi. Small fragments ofa clot, emboli, may detach from the body of the clot and travel throughthe circulatory system to lodge in distant organs and initiate furtherclot formation. Heart attack, stroke, renal and pulmonary infarcts arewell known consequences of thromboembolic phenomena.

A blood clot is a gelled network of protein molecules within which aretrapped circulating blood cells, platelets, and plasma proteins. A majorprotein component of a clot is fibrin, which forms a relativelyinsoluble network in the clot. Proteolytic enzymes, particularlyfibrinolytic enzymes, have been used to dissolve vascular obstructions,since disruption of the fibrin matrix results in dissolution of theclot. Clots are formed when soluble fibrinogen, which is present in highconcentrations in blood, is converted to insoluble fibrin by the actionof thrombin. Fibrinolytic enzymes dissolve the fibrin matrix of a clot,and fibrinogenolytic enzymes digest the fibrin precursor fibrinogen.

Intravascular clots may be removed after their appearance by means ofenzymes capable of dissolving fibrin (fibrinolytic enzymes); and theprobability of clot formation can be reduced by lowering theconcentration of circulating fibrinogen, using enzymes that degradefibrinogen (fibrinogenolytic enzymes).

Plasmin, a naturally-occurring fibrinolytic and fibrinogenolytic enzyme,is relatively unstable in the human circulatory system and, therefore,circulates primarily in its more stable inactive form, plasminogen. Theactivation of plasminogen to plasmin occurs by cleavage of a singlearginyl-valine bond and is catalyzed by plasminogen activators.Urokinase and tissue plasminogen activator activate plasminogen bydirect cleavage of the arginyl-valine bond. Streptokinase andstaphylokinase are plasminogen activators of bacterial origin thatactivate plasminogen indirectly by forming a complex with plasminogen;this streptokinase-plasminogen complex behaves as a plasminogenactivator that activates other plasminogen molecules by cleaving thearginyl-valine bond.

Thromboembolytic therapies have involved the administration of aplasminogen activator; e.g., the direct intravenous injection of aplasminogen activator alone, the reinjection of a patient's plasma towhich a plasminogen activator has been added ex vivo, the injection ofplasma protein fractions previously mixed with streptokinase, or theinjection of a preparation of porcine plasmin stabilized with addedlysine in conjunction with streptokinase.

Fibrinogenolytic therapy aimed at reducing the risk of thrombosis hasinvolved injection of snake venoms, e.g., Angkistrodon rhodostoma, whichcontain enzymes that degrade fibrinogen.

SUMMARY OF THE INVENTION

The invention features a fibrinolysis or fibrinogenolysis treatmentwhich includes the parenteral introduction of human or mammalian plasminor mini- or micro-plasmin into the body of a patient, the plasmin beingin fibrinolytically/fibrinogenolytically active form, in an amount andfor a time sufficient to permit the active plasmin to reach aconcentration in the patient's bloodstream sufficient at least to reducecirculating fibrinogen levels.

In preferred embodiments, the amount of active plasmin and the durationof treatment permits the active plasmin to reach a concentration aboutthe site of an intravascular clot sufficient to lyse the clot. Inaddition, or alternatively, the amount of active plasmin and theduration of treatment permits the active plasmin to reach aconcentration sufficient to digest circulating fibrinogen at a ratesufficient to prevent the formation of a blood clot.

In preferred embodiments, fibrinolytically/fibrinogenolyticallyplasminogen, or one or a mixture of its active analogs, is converted tofibrinolytically/fibrinogenolytically active plasmin, or thecorresponding analog, extracorporeally (i.e., outside of the body),preferably by exposure to a physically contained, immobilizedplasminogen activator, e.g., urokinase or tissue plasminogen activatoror active analogs thereof, prior to the process of introducing theplasmin into the human body. Preferably, the plasminogen activator iscovalently bonded to a matrix that may be, for example, a porous polymermembrane, e.g., nylon.

As used herein, "physically contained" plasminogen activator means theactivator is insolublilized, entrapped, or encapsulated; "immobilizedplasminogen activator" means that the plasminogen activator ismatrix-bound, carrier-bound, or support-bound; a "physically contained"or "immobilized" plasminogen activator is prevented from accompanyingthe active plasmin into the body; "parenteral introduction" of a drugmeans the drug is introduced to the body other than by way of thegastrointestinal tract, e.g., intravenously, intraarterially,intraperitoneally, subcutaneously, intraocularly, or inhalationally;"extracorporeal" administration means from a point outside of thepatient's body; and "substantially coincident" means either during theprocess of, i.e., at the same time as, or immediately prior tointroducing the drug into the patient's body. The time period betweenthe conversion of plasminogen to plasmin and injection of the plasmininto the body will typically be a period of 10 minutes, preferably 5minutes, and most preferably less than 1 minute. The term "immediatelyprior to" means that the plasminogen to plasmin conversion and theintroduction of plasmin into the patient occur close enough in time suchthat the active plasmin maintains at least 80%, preferably 90-99%,activity during that intervening time period. Preferably, human plasminmade according to the invention is substantially free of elements thatinterfere with its clot-lysing ability; i.e. it may be 98-99% pureplasmin, except for the presence of plasminogen in the plasmin sample.

As used herein, "clot lysis" refers to the partial or completedissolution of a clot. As used herein, "mini-plasmin" or"mini-plasminogen" refers to that form of plasmin or plasminogen whichcontains, in addition to the enzymatic (i.e., catalytic) domain of themolecule, a single kringle; "micro-plasmin" or "micro-plasminogen"refers to a truncated form of plasmin or plasminogen which lacks allfive kringles and the amino-terminal domain (i.e, preceding the firstkringle); "mammalian" plasmin refers to both human and non-humanplasmins, the non-human forms include but are not limited to bovine,porcine, or ovine plasmins.

In other preferred embodiments, fibrinolytically/fibrinogenolyticallyinactivated plasmid (inhibited plasmin), or an inactive analog thereof,is converted to active plasmin or its analog extracorporeally, byexposure to a substance capable of removing an inhibitor and/orstabilizer of the active plasmin, wherein the removal results in plasminreactivation. The inhibitor preferably includes lauryl sulfate orsimilar hydrophobic anions or cations, which reversibly inhibit theenzymatic activity of plasmin. The inhibitor removing substance mayinclude an adsorptive matrix or an ion exchange resin.

Analogs of plasminogen or plasmin include but are not limited to glu-,lys-, or mini- or micro-plasmin or any amino acid sequence having atleast 70% homology with plasminogen or plasmin or their truncated formsand possessing the enzymatic properties of either molecule. Plasminogenmay be purified natural plasminogen, or may be chemically synthesized orexpressed from recombinant DNA. Generally, the invention is unlimitedwith respect to the type of plasmin active substance employed, be itnatural form, truncated, or an analog of plasmin, and for the purpose ofthe invention, all such forms are considered in material respects to beembraced within the term "plasmin". Analogs of urokinase or tissueplasminogen activator include those proteins having amino acidinsertions, substitutions, or deletions which result in a moleculehaving plasminogen activating activity; such analogs would includetruncated forms of these molecules.

Another aspect of the invention features a stabilized enzymaticallyinactive plasmin composition including hydrophobic ions comprisingbranched or straight chained alkyl groups having from about eight toabout 20 carbon atoms linked to an anion group, preferably a sulfurcontaining anionic group such as sulfate, a cationic group such asquaternary ammonium, or both. The currently preferred hydrophobic ionsare lauryl sulfate ions. These compositions are characterized by theability to be reactivated upon removal of the ions by, for example, anappropriate ion exchange medium.

Fibrinolysis or fibrinogenolysis treatment according to the invention isuseful to treat or prevent heart attack, stroke, and thromboembolicvascular occlusion, with or without associated organ infarction. Therisk of developing a thrombus can also be reduced according to theinvention, e.g., for diabetics, who have an increased risk of developingthrombi, or for pregnant women or women who use oral contraceptives, whohave an increased risk of thrombophlebitis. The amount of plasminogenactivator required for fibrinolysis treatment according to the inventionis a small fraction of that required for plasminogen activator injectiontherapy to dissolve blood clots, and that small fraction may be used anumber of times. In addition, since the plasminogen activator remainsextracorporeal during treatment, it will escape the rapid physiologicalturnover to which plasminogen activators are normally subjected in thecirculatory system; likewise, it will also avoid contact with thepatient's immune system, and thus the patient will not mount an immuneresponse to it. Consequently, plasminogen activators used in accordancewith the invention may be derived from human as well as non-humansources.

Administration of active human plasmin according to the invention is,therefore, simple, economical, adaptable to individual patient needs,and does not require the use of foreign proteins or drugs which may inthemselves have unwanted side-effects, e.g., an immune reaction.Administration of mammalian mini- or micro-plasmin will also have theseadvantages and, due to the truncated form of these molecules,potentially will not raise a significant immune response whenadministered to a patient. The administration of non-human mammaliantruncated plasmins can be performed according to the invention becausethese truncated versions of plasmin contain little more than one-third(i.e., the active region) of the plasmin molecule, and thereforepresumably present fewer foreign epitopes for the human immune system torespond to.

The treatment of thromboembolism according to the present invention issuitable for acute episodes requiring short-term treatment or for morelong-term, continuous or intermittent courses of treatment.Pharmacological variables associated with introduction of drugs into thebody, e.g., the rate of inhibition or inactivation of or the removal ofplasminogen activators from circulation, do not play a role inthrombolytic therapy according to the invention since the plasminogenactivator remains extracorporeal. In addition, individual patient needscan be accommodated by administering a precise amount of plasmin to thepatient that has been adjusted to individual factors, e.g., the amountof circulating inhibitors of plasmin.

The course of therapy can be monitored, if desired, e.g., by repeatedestimation of the patient's circulating inhibitor levels, duringtreatment periods of any duration, thereby permitting the dynamicadjustment of therapy. In therapies based on injected plasminogenactivators a large fraction of the patient's plasminogen is consumed,thereby limiting the extent, duration and frequency of therapy. Intherapy according to the invention there is no depletion of patient'splasminogen, which remains available; and the extracorporeal reservoirsof both plasminogen and activators are unlimited, permitting treatmentsof unrestricted frequency and duration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing hydrolysis of TAME substrate in the presenceof membrane-bound enzyme.

FIG. 1B is a graph showing hydrolysis of KABI S-2160 substrate in thepresence of membrane-bound enzyme.

FIG. 2 is an SDS polyacrylamide gel of mini-plasminogen activated bymembrane-bound urokinase.

FIG. 3 is a graph of radioactivity levels recorded from a monitoroverlying a radioactive blood clot in a dog.

DESCRIPTION

A patient suffering from vascular obstruction, e.g., a victim of heartattack, stroke, or renal or pulmonary infarction, or at high risk ofdeveloping vascular obstruction or peripheral vascular disease, may betreated according to the invention by administration ofextracorporeally-activated or reactivated plasmin to the patient. Theextracorporeal activation of plasminogen is achieved by immobilizing aplasminogen activator on a matrix having a large surface area, e.g., aporous membrane, and perfusing the membrane with a solution of humanplasminogen in its native or truncated forms. The membrane is mounted ina device close to the site of injection. Plasminogen is activated uponencountering the immobilized plasminogen activator while traversing themembrane, and the plasmin formed passes immediately into thebloodstream.

The invention is based primarily on the realization that fibrinolyticand fibrinogenolytic therapy can be conducted effectively and can becontrolled by direct infusion of active forms of plasmin despiteplasmin's notorious instability and propensity for autodigestion.Broadly, this is accomplished by treating a stabilized plasminpreparation to remove. stabilizing moieties, or catalytically orotherwise producing active plasmin from a plasmin precursor such asplasminogen, just prior to parenteral infusion. The means by which theinfusable, active plasmin is produced at the treatment site does not perse comprise an aspect of this invention, except as set forth in theclaims. Currently preferred devices for implementing the invention aredisclosed herein to enable those skilled in the art to make and use theprocesses of this invention. For further particulars of apparatussuitable for use in this invention, see copending application Ser. No.07/750,920, filed on the same day as this application, the disclosure ofwhich is incorporated herein by reference. As noted above, purifiedplasmin is chemically unstable due to its susceptibility to proteases,and its tendency to self-digest. Included in the invention is thediscovery that certain hydrophobic ions inhibit the autolytic activityof plasmin. Lauryl sulfate ions are inhibitors of plasmin when presentin amounts, depending on protein concentration, equal to or greater than0.05%. Other hydrophobic anions or cations which may have the sameeffect and are separable readily are sarcosyl, deoxycholate, andcetyltrimethyl ammonium halides. Generally, useful hydrophobic ionscomprise moieties having 8 to 20 carbon atoms attached to one or moreionic groups such as sulfate, sulfonate, phosphate, or quaternaryammonium. The invention thus also includes the extracorporealreactivation of inactive plasmin; e.g., a mixture of plasmin and areversible inhibitor of plasmin, e.g., lauryl sulfate, which may bereactivated extracorporeally by removal of the inhibitor just prior toinjection of the plasmin using, for example, an ion exchange resin.

Fibrinolysis and fibrinogenolysis treatment according to the inventionis described below, including preparation of immobilized plasminogenactivator and substrate plasminogen or substrate derivatives,preparation of a stable mixture of inactive plasmin, the reactivation ofinactive plasmin, and fibrinolysis treatment in vivo by direct injectionof purified active plasmin.

Preparation of Immobilized Plasminogen Activator

Immobilized plasminogen activator may be prepared using a supportconsisting of nylon membrane, of 1-3μ average pore size, (PallCorporation, Glen Cove, N.Y.). In its preferred version, such a membranebears a high density of unsubstituted carboxylate groups (Pall.No.BNPCH5 or BNNCH5), which act as starting points for chemicalmodifications that allow anchoring of proteins.

Nylon membrane sheets are cut into the shape of discs of desireddiameter and derivatized as follows. A solution of 0.5M sperminetetrahydrocholoride in water is brought to pH 7.0-7.1 by the carefuladdition of NaOH; separately, a solution of a water solublecarbodiimide, preferably 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDAC), (0.5 M in water) is brought to pH 5.0-5.05 by theaddition of dilute hydrochloric acid; the two solutions are mixed inequal amounts and membrane discs are immersed in the mixture andincubated overnight at room temperature. The discs are washed copiouslyfirst with distilled water and then with 1.0M NaHCO₃. The discs are thenpacked in solid, finely-pulverized succinic anhydride (500 mg per cm²disc surface area), and sufficient dipotassium hydrogen phosphate (0.5M) is added to thoroughly irrigate the disc and succinic anhydridepacking (ca. 0.3-0.5 ml/cm² disc area). The reaction is allowed toproceed overnight at room temperature. Small sample discs areincorporated alongside the membranes being treated and tested for thepresence of residual free amino groups. This succinylation procedure canbe repeated, if necessary. The succinylated discs are rinsed free ofprecipitated succinate, washed under suction first with NaHCO₃ (0.5-1.0M) and then with water, and dried. The discs may be stored for months atroom temperature, with no change in properties.

The dried membrane filter discs are mounted and securely clamped inholders that permit them to be perfused, and the entire assembly isincorporated into a circuit, driven by a peristaltic pump, in which thediscs are continuously perfused for at least 1 hour at >30° C. with asolution of N-hydroxysuccinimide and EDAC, both at 50 mM, in puretert-butanol. At the end of the perfusion, the mounted discs areperfused briefly with 1 mM HCl at room temperature, blotted, swirled inice-cold distilled water for a few minutes, then placed in a shallowdish, previously rinsed with 0.5% detergent TRITON X-100 in water, whosediameter is just sufficient to accommodate the membrane discs. Thecarboxylate groups have now been activated for protein coupling.

Highly purified human urinary urokinase is coupled to the membrane discsimmediately after activation as follows. Lyophilized urokinase, e.g.WINTINASE (Winthrop Laboratories, Division of Sterling Drug, Inc., NewYork, N.Y.), UKIDAN (Serono, Autonne, Switzerland) is dissolved in 10 mMHEPES, pH 7.0-7.4 at a concentration of 4-5 mg per ml. Sufficientsolution is pipetted into small shallow dishes so as to thoroughlyimpregnate the activated membranes, e.g., 15-20 μl/1 cm² of membrane. Atotal of approximately 400 μg or 1.2 mg urokinase is used to impregnatea membrane of 25 mm or 47 mm diameter, respectively. After immersing themembranes in urokinase solution, the dishes are sealed, and incubated ina moist chamber at 4° C. for 14-16 hours, preferably with gentle rockingagitation. The coupled membrane is rinsed with 1 mM HCl by low speedcentrifugation in a polypropylene tube, and the rinsing fluid collected,pooled with residual incubation medium, and assayed for remainingnon-membrane-bound urokinase.

Assay of Membrane Bound Urokinase

Membrane bound enzyme is assayed by pumping substrate, either small andsynthetic, or macromolecular protein substrates (plasminogens), throughthe membrane; the former measures the amount of active enzyme bound,whereas the latter yields an estimate of the catalytic capacity of themembrane in plasminogen activation. The membrane to be assayed ismounted in a filter holder (for example, Millipore Nos. SX0002500 andSX0004700 for 25 mm and 47 mm diameter, respectively), which isconnected to a peristaltic pump. Temperature control may be achieved byimmersing the substrate reservoir and connecting tubing in a temperatureregulated bath. The substrate solution is pumped through the membrane,or several membranes assembled in series, e.g., 1 ml aliquots of theeffluent are taken. The absorbance change in the effluent compared withthe substrate solution gives the concentration of product which,multiplied by the flow rate, yields the activity in terms of moles perunit time for small substrates;, assay of plasmin using KABI S-2251 isused for estimating the rate of plasminogen activation. Given the valueof apparent K_(cat), which is derived from measurements of enzymeactivity in free solution, the estimate of active bound enzyme is easilyobtained from the observed rate of product formation. In practice, assayof bound enzyme activity should be made under conditions of perfusion inwhich no more than 10% of small substrate are hydrolysed; flow rates of10-15 ml/cm² /h give maximum apparent rates of substrate hydrolysis.

The small substrates dissolved in Tris-HCl buffer (0.1 M, pH 8.8) areeither TAME (tosyl-L-arginine methylester, 10 mM) or KABI S-2160(N-benzoyl-phe-val-arg-p-nitroanilide, 0.2 mM). The hydrolysis of TAMEis measured by change in absorbance at 247 nm, and that of KABI S-2160at 405 nm. An illustration of the results of such an assay is given inFIG. 1.

Preparation of Substrate Plasminogen

Human plasminogen is prepared at 4° C. according to a modified procedureof Liu et. al., Canadian J. Biochem. 49:1059-1061 (1971). Five hundredgrams of frozen Cohn fraction III or II & III paste is pulverized at 4°C. using a mortar and pestle, then added in portions with constantstirring to 5 liters of phosphate buffered saline (PBS), containing 1 μMp-nitrophenyl-p-guanidinobenzoate. Stirring is continued for 4-5 hours,until the paste is fully and evenly suspended. The solution is thencentrifuged at 12000×g for 20 minutes at 4° C., and the gelatinouspellet discarded. The supernatant is filtered under gravity through"fast" filter paper, then brought to 10% of saturation (e.g. 50 g/l), bythe addition of solid ammonium sulfate, and centrifuged once again at12000×g for 20 minutes at 4° C. The resulting pellet is discarded, andthe lipid-like material floating on the supernatant removed byfiltration through a gauze plug.

The filtered supernatant is then pumped, e.g., at 600-900 ml per hour,into e.g., a 230 ml, 4.8×15cm column packed with G-15 SEPHADEX in PBS,and the outflow passed directly into a second column, of, e.g., 750-800ml volume and 10 cm. in diameter, packed with lysine-agarose (Pharmacia4B or 6B) and pre-equilibrated with PBS. The entire system is washedwith PBS (e.g., 250 ml), the G-15 column disconnected, and thelysine-agarose column is washed with an additional 1.5 column volumes ofPBS until the A₂₈₀ drops below 0.15. The column is then washed with 1column volume of a solution consisting of 4 parts of ethylene glycol and6 parts of potassium phosphate buffer (0.5 M, pH 8.0), followed by onecolumn volume of PBS. Plasminogen is then eluted from the column with alinear gradient (2.5-3 column volumes) of epsilon aminocaproic acid(0-25 mM) in PBS, and collected in fractions. The fractions having thehighest concentration of protein are pooled and precipitated at 50%saturated ammonium sulfate in the presence of benzamidine (50 mM), thepH being kept near neutrality by periodic addition of small volumes oftris-hydroxymethyl aminomethane (tris) base (1M).

The precipitated plasminogen can be stored under 50% saturated ammoniumsulfate containing 50 mM benzamidine for many months with no loss ofactivity. After desalting and redissolving in PBS, it can be useddirectly for generating plasmin as described below.

Preparation of Substrate Mini-plasminogen

Mini-plasminogen is prepared according to a modified procedure of Powellet. al., J. Biol. Chem. 225:5329-5335 (1980). The plasminogenprecipitate, e.g., 300 mg, is suspended in ammonium sulfate-benzamidine,as described above, centrifuged at 10,000×g for 30 minutes at 4° C. andthe resulting supernatant discarded. The pellet is dissolved in aminimum volume e.g., 15-20 ml, of 100 mM NaCl-50 mM Tris, pH 8.0, at 4°C. and desalted by passage through a 230 ml, 4.8×15 cm column of G15SEPHADEX in the cold. Protein-containing fractions eluted from thecolumn are pooled and diluted at room temperature with startingNaCl-Tris buffer to 3 mg/ml. 20,000 Kallikrein inhibitor units ofaprotinin, and 1.7 mg pancreatic elastase are then added to the pooledprotein fractions and the solution is incubated at room temperature withgentle stirring for 5 hours. The reaction is terminated by addition ofmethoxysuccinyl-(-ala-ala-pro-val) chloromethylketone to 10⁻⁴ M, andstirred for a further 30 minutes. The solution is dialyzed overnight at4° C. against a large volume of 0.1 M sodium phosphate buffer, pH 8.0,using tubing with molecular weight cutoff at 6500 daltons. 300 mg ofdialyzed plasminogen in solution is applied to a 4.8×15 cm, 230 mllysine-agarose column equilibrated in 0.3 M sodium phosphate buffer, pH8.0, and mini-plasminogen is eluted in 300 ml of the same buffer.Protein-containing fractions are pooled, benzamidine added to a finalconcentration of 50 mM, and miniplasminogen precipitated by the additionof solid ammonium sulphate in several portions to a final concentrationof 80% saturation.

The macromolecular substrates glu- and lys-plasminogen as well astruncated forms such as mini- and/or micro-plasminogen are dissolved in90 mM NaCl, 5 mM NaPO₄, pH 7.3-7.5, 1.8% dextrose, usually at aconcentration of about 30 μM. Glu-plasminogen is the naturally occurringform of plasminogen in circulation; the N-terminal amino acid residue isglutamic acid. Lys-plasminogen, having an N-terminal lysine residue, isderived from glu-plasminogen by limited proteolysis, usually catalyzedby plasmin, whereby a peptide fragement 77 residues long is cleaved fromthe amino terminal domain. Mini-plasminogen is derived from either glu-or lys-plasminogen by limited proteolysis, catalyzed by pancreaticelastase, whereby a fragment consisting of the proenzyme domain ofplasminogen with a single attached kringle is generated, the remaining 4kringles and intervening peptides having been separated.(Sottrup-Jensen, L. et. al., Prog. in Chemical Fibroinolysis andThrombosis 3:191-209 (1978) Davison, J. et. al., Eds., Raven Press,N.Y.). Microplasminogen consists of the proenzyme domain of plasminogenwith a stretch of connecting peptide and a few residues of kringle 5attached at its N-terminal end; it is generated by the action of plasminon plasminogen (Shi, G.-Y., and Wu, H.-L., J. Biol. Chem., 263:17071-5(1980).

The rate of plasminogen activation, as well as the fraction that isactivated to plasmin are influenced by numerous factors, includingplasminogen concentration, flow rate, membrane area, enzyme binding areawithin the reaction zone, and numbers of membranes in series within thereaction zone. These parameters can be adjusted to achieve any desiredtherapeutic goal in terms of plasmin formed per unit time for anyfraction of plasminogen activated. In a typical run, two 47 mm membranesmounted in series will activate approximately 80% of the perfusedmini-plasminogen at a concentration of 30 μM and a flow rate of 70ml/hour, yielding about 1.7 μmoles of mini-plasmin/hour. Membranes canfunction continuously at constant rates for at least 3 hours.

When membrane coupling is performed, 80% of available urokinase isremoved from solution and bound to the membrane. Of this amount, 20-25%is catalytically active in hydrolysis of small substrates, andapproximately 5% is active in plasminogen activation.

In FIG. 2, samples were run on an SDS-polyacrylamide gel with2-mercaptoethanol (121/2% acrylamide). Lane two containsmini-plasminogen, whose molecular weight is somewhat less than that ofovalbumin. In this preparation, mini-plasminogen appears as two closelyspaced bands. Lanes three to six contain sequential samples afterperfusion of mini-plasminogen through porous nylon membranes containingimmobilized urokinase. The perfusion rate for samples in lanes three tofive was about 70 ml/hr. The major band from mini-plasmin is the light(or B) chain, at a molecular weight just below that of carbonicanhydrase; the amino terminal fragments of mini-plasmin appear as adoublet between soybean trypsin inhibitor and lysozyme. For the samplein lane six, the perfusion rate was reduced to about 35 ml/hr; theproportion of unactivated mini-plasminogen is decreased relative tolanes three to five, and the proportion of plasmin light chain isincreased. Lanes one and seven contain molecular weight referencestandards. These are (from top to bottom): phosphorylase b 97,400daltons; bovine serum albumin 66,200; ovalbumin 45,000; carbonicanhydrase 31,000; soybean trypsin inhibitor 21,500; and lysozyme 14,400.

Preparation of Stored Inactive Plasmin

A second fibrinolytic treatment according to the invention also involvesthe direct infusion of highly purified plasmin. This plasmin isprepared, stored, and formulated in advance. Plasmin exhibits, like anyproteases, a strong tendency to self-digestion, especially under theconditions of high concentration that are encountered during itspreparation, storage and formulation for delivery. It is desirable toprevent autolysis in order to preserve catalytic activity. A preferredway of accomplishing such suppression is by the addition of suitableconcentrations of lauryl sulfate ions, in the form of sodium laurylsulfate, which inhibits plasmin-catalyzed proteolysis. However, laurylsulfate can be toxic and, therefore, must be completely removed from theplasmin preparation prior to injection. This is convenientlyaccomplished by passing the solution of plasmin and lauryl sulfate overa matrix capable of removing lauryl sulfate either by adsorption and/orion exchange. Thus, the plasmin is activated coincident with itsinjection into the patient's body.

All operations are performed under sterile conditions using sterile,pyrogen-free reagents. Plasmin, or one of its truncated forms (mini- ormicro-plasmin) is produced by perfusing the corresponding plasminogenthrough one or more urokinase membranes, prepared as described above.The flow rate, plasminogen concentration, temperature and numbers ofmembranes in series are selected to activate at least 95% of theperfused plasminogen to plasmin; for example, two 47 mm membranesperfused with 30 μM miniplasminogen at approximately 50 ml/hr and 25° C.The effluent is collected directly into a chilled vessel containing asolution of sodium lauryl sulfate sufficient to yield a final sodiumlauryl sulfate concentration of 0.05-1.0%, e.g., a solution of 0.5-10%sodium lauryl sulfate in a volume of buffered H₂ O one tenth that of theanticipated final volume of effluent to be produced. Alternatively, toachieve a greater proportion of plasmin in the final product, theeffluent may be led through a refrigeration bath and directly onto acolumn bed of immobilized plasmin inhibitor (e.g. aprotinin), where itis bound in an inhibited state; the material emerging from this bedcontains the remaining, unactivated plasminogen which may be recycledonto the urokinase membrane to achieve a substantially completeconversion to plasmin.

At the termination of plasminogen activation, any residual unactivatedplasminogen is removed by washing the column bed with PBS buffer, andthe plasmin product is recovered by elution with 90 mM NaCl-1 mM HCl,and collected into a buffered solution of sodium lauryl sulfate,designed to neutralize the HCl, as indicated above. The inactive plasminis concentrated by the addition of solid ammonium sulfate to 80% ofsaturation, the precipitated plasmin is collected by centrifugation, theammonium sulfate removed by dialysis against large volumes of watercontaining sodium lauryl sulfate (0.1%) and 90 mM NaCl, and the dialyzedplasmin solution lyophilized. For therapeutic administration, theinactive plasmin is reconstituted by addition of sterile, pyrogen-freewater containing 1.8% of dextrose, 90 mM NaCl and 0.1% lauryl sulfate,final concentrations. The inactive plasmin solution is then perfusedthrough a matrix capable of retaining lauryl sulfate ions and thusremoving them from the plasmin preparation. For example, the inactiveplasmin solution may be pumped through a column of EXTRACTIGEL (PierceChemical Co., Rockford, Ill.), an adsorbing matrix, at a rate notexceeding 1 ml per cm² per minute. The lauryl sulfate ions are thusretained by the matrix and reactivated plasmin is produced.

Alternatively, the lauryl sulfate-stabilized plasmin may be activated bycontacting it with a plurality of ion exchange resin particles. Theparticles may vary in size, e.g., from 10-350 mesh, and the resin may beany conventional material capable of adsorbing lauryl sulfate ions,e.g., AG11 A8 (Bio-Rad Laboratories, Richmond, Calif.).

Experimental Fibrinolysis

The fibrinolysis treatment of the invention, in which human plasmin isdirectly administered to a patient, can be tested in vivo by introducinga radioactive clot into, e.g., the external jugular vein of a dog, andinjecting plasmin using the apparatus of the invention into the dog'scirculatory system. Dissolution of the clot may be followed bymonitoring the level of radioactivity; a decrease in the level ofradioactivity at the site of the clot indicates dissolution of the clot.

Testing was performed using a plasmin preparation of the invention. Aclot was prepared using freshly-drawn, anticoagulated and thenrecalcified whole human blood to which a small amount of ¹²⁵ I-labeled,highly purified human fibrinogen has been added. The clot is secured ina stainless steel coil that is introduced into and lodged in theexternal jugular vein; the use of radioactive clots prepared ex vivo haspreviously been described by Matsuo et. al., (Nature 291:590 (1981)).The clot is introduced into healthy, well-conditioned male hounds, atleast 20 kg in weight, which have been fasted overnight and prepared forsurgery under standard conditions. One side of the neck, and theipsilateral fore and hind legs are shaved, catheters are placed in therespective cephalic and saphenous veins, and the animals anesthetized bythe intravenous administration of thiamylal sodium (20 mg/kg). Anintratracheal tube is then inserted, and the animals maintained oninhalational anesthesia consisting of nitrous oxide:oxygen 2:1containing 1-2% halothane. Physiological saline is dripped through bothcatheters at a slow maintenance rate (2-3 drops per minute). The skinoverlying the external jugular vein is incised (2-3 inches) parallel tothe vein, and the internal and external maxillary veins exposed by bluntdissection for about 2 inches before their confluence; a similarexposure is practiced for the external jugular vein. The internalmaxillary vein is ligated, anteriorly, leaving an accessible length ofabout 2 inches before the confluence, the external maxillary andexternal jugular veins are clamped, and the clot is set in place. Theclamps are removed to reestablish blood flow, a gamma detector is fixeddirectly above, <5 mm from the surface of the vein, and theradioactivity monitored at 10-20 minute intervals. Twourokinase-membrane filters in series are connected to the catheter inthe cephalic vein and mini-plasminogen (30 μM) dissolved in 90 mL NaCl,10 mM Na phosphate pH 7.3-7.4, 1.8% dextrose, is pumped through at arate of 70 ml/hr.

The radioactivity level registered by the detector is recorded as afunction of time after onset of the mini-plasmin infusion. A decrease inradioactivity at the site of the clot indicates dissolution of thefibrin clot. Six separate experiments using dogs fitted with intravenousclots were performed, and the results demonstrated essentially completeclot lysis in reproducible time periods in each experiment. FIG. 3 is agraph of results in which radioactivity levels were recorded from amonitor overlying a radioactive blood clot inserted in the externaljugular vein of a dog. Radioactivity is recorded as a function of timefollowing the onset of infusion of mini-plasmin which was generated byperfusing mini-plasminogen through a nylon membrane containingimmobilized human urokinase.

Blood samples are aspirated from the saphenous vein at timed intervals,and are assessed for fibrinogen content by determining clottability,either spontaneous or following addition of human thrombin (0.25 unitper ml, final concentration), aprotinin having been added to block anyplasmin activity. These determinations reveal that fibrinogen decreasesprogressively from the onset of the infusion, its removal beingessentially complete in about one hour under these conditions.

Other fibrinolytic treatments, which involve a chosen plasminogenactivator and a given preparation of plasminogen may also be tested inthe a system described above. Parameters such as concentration of activeplasmin administered, duration of treatment, type of plasminogenactivator, and type of membrane, etc., may be varied, and thesevariations may also be tested in such a canine system.

Dosage and Use

Methods of the invention include fibrinolytic/fibrinogenolytictreatments, the dissolution of intravascular thrombi, and reduction ofthe risk of thrombus formation. Persons at risk for thrombus formationinclude but are not limited to diabetics and pregnant women. Diabeticscarry a higher than normal level of fibrinogen and, therefore, have ahigher risk of developing thrombi. The administration of plasminprophylactically to a diabetic would lower fibrinogen levels and thusreduce the risk of clot formation.

The duration of fibrinolytic and/or fibrinogenolytic treatment accordingto the invention may vary from a short single dose administration ofplasmin, e.g., 1-30 μmoles of plasmin for a 150 lb. person within a 6hour period to much larger quantities during continuous or intermittentadministration for days to weeks, depending upon the size and locationof the clot. For example, if the clot is venous, the duration oftreatment may be days, whereas if the clot is arterial, only hours oftreatment may be required. Short, single dose treatments may be requiredfor conditions such as myocardial infarction; longer thrombolyticregimens for thrombophlebitis and pulmonary embolism; and prolonged,continuous and/or intermittent treatment may be used to treat coronaryocclusion and other conditions for which prophylactic therapy may bedesirable, e.g., to reduce the risk of clot formation. Continuoustreatment includes the uninterrupted administration of plasmin;intermittent therapy includes the administration of plasmin which isinterrupted by minutes, days, or weeks. A longer thrombolytic orfibrinolytic/fibrinogenolytic therapy will require adjustment dependingupon the ultimate desired level of circulating fibrinogen. If a smallreduction in fibrinogen concentration is required, more frequentinjections of low doses may be needed to maintain a given depression ofthe fibrinogin level.

Clot dissolution reflects the fibrinolytic action of plasmin, and theduration and effectiveness of thrombolytic therapy followingadministration of plasmin depend primarily on the balance between therates of plasmin introduction, and plasmin removal and/or inhibition bythe plasmin inhibitor, ∞2-antiplasmin, or other inhibitors of plasmin.Factors to be taken into account when adjusting plasmin dosage for clotdissolution include physical factors such as height, weight, and age ofthe patient; the location of the blood clot, and circulating levels ofplasmin inhibitors, such as ∞2-antiplasmin and ∞2-macroglobulin.∞2-antiplasmin, which has a normal range of plasma concentration in vivoof approximately 1 μM±20%, is ordinarily the dominant factor regulatingplasmin action in the circulation; plasmin combines irreversibly with∞2-antiplasmin to form a 1:1 complex and is thereby inhibited before itcan attack clots or other proteins. The level of circulating∞2-antiplasmin is important in assessing plasmin dosage, and∞2-antiplasmin must be titrated to a level at which no more than 15% ofthe normal circulating concentration is present. A high initial level of∞2-antiplasmin will require a large dose of extracorporeal plasmin to beadministered parenterally.

Other Embodiments

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

Other embodiments of the invention are within the following claims.

What is claimed is:
 1. An inhibited plasmin composition comprising aplasmin preparation stabilized with lauryl sulfate, wherein saidinhibited plasmin composition is fibrinolytically andfibrinogenolytically inactive and fibrinolytically andfibrinogenolytically reactivated upon removal of said lauryl sulfate. 2.The composition of claim 1 wherein said lauryl sulfate is present in anamount between about 0.05% and about 0.10% by weight.
 3. A stabilizedplasmin composition comprising a plasmin preparation in combination withlauryl sulfate, said lauryl sulfate being present in said composition inan amount sufficient to inhibit said plasmin from auto-digestion,whereupon removal of said lauryl sulfate from said composition resultsin plasmin having auto-digestion activity.